During infection, different classes of bacteriophage T4 genes are transcribed by the host RNA polymerase, which is modified by the phage at middle and late times. Transcription from middle promoters also requires the T4 MotA protein. We have been investigating the mechanism of MotA at the T4 middle promoter PuvsX. Like other motA-dependent promoters, PuvsX contains a match to the sequence (t/a) (t/a) TGCTT (t/c) A ('MotA box'), centered at -30 relative to the start of transcription. In addition, other possible MotA boxes are found in PuvsX, centered at -35, -51, -70, and -87. Our DNase I footprint analyses indicate that MotA protects PuvsX in the region from -25 to -59. This includes the MotA box at -30, but also the farther upstream sites centered at -35 and -51. In vitro transcription of a template lacking the MotA box at -51 indicates that while this site is not required for activation, it contributes to MotA action under less favorable transcription conditions. The first 8 amino acids (aa) of MotA are replaced with 11 different aa in the MotA mutant protein Mot21. Although Mot21 binds PuvsX similarly to wild type MotA, it does not activate transcription. In addition, C-terminal fragments of MotA, starting at aa 102 or aa 105, also bind PuvsX DNA. These results identify the C-terminal half of the protein as the DNA binding domain and suggest that the N-terminal half may be involved in protein-protein interactions. The T4 segA gene encodes a Mg++-dependent endonuclease, which is related by sequence to endonucleases present in group I introns. Our characterization of the SegA cleavage activity indicates that although SegA prefers to cleave at certain sites, as the concentration of protein or incubation time is increased, any DNA is digested. Linear cleavage products are ligated by T4 DNA ligase, and SegA activity is stimulated by the addition of ATP. Thus, SegA exhibits a hierarchy of site preference, and the apparent specificity of SegA is governed by the level of the protein, the length of incubation, and the presence of any factors that can stimulate or reverse the reaction.